CN111040995A - Method for amplifying tumor killer T cells in tumor infiltrating lymphocytes - Google Patents
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Abstract
The present disclosure relates to a method for amplifying tumor killer T cells in tumor infiltrating lymphocytes, comprising the steps of: a. mixing the monocyte suspension with the feed layer material suspension, and performing suspension culture for 96-120h to obtain a first culture; wherein the feeding layer material comprises 3D tumor organoids after irradiation inactivation; b. mixing the first culture with a tumor infiltrating lymphocyte culture medium, and performing suspension culture for 65-72h to obtain a second culture; c. mixing the second culture with a tumor infiltrating lymphocyte culture medium, and performing suspension culture for 72-96h to obtain a third culture; d. removing the feeder layer material in the third culture, and separating to obtain the tumor killing T cells. By adopting the technical scheme, the in-vitro directional amplification of the tumor killing T cells can be realized, the amplification efficiency and the tumor killing activity of the tumor killing T cells are greatly improved, and the consistency of the amplification efficiency among different amplification batches can be improved.
Description
Technical Field
The disclosure relates to the technical field of biological medicines, in particular to a method for amplifying tumor killer T cells in tumor infiltrating lymphocytes.
Background
Tumor-infiltrating lymphocytes (TILs) are a heterogeneous lymphocyte population mainly comprising lymphocytes existing in tumor interstitium, and are characterized in that T Cell Receptors (TCRs) are expressed, T lymphocytes are mainly comprising T lymphocytes, and a small part of the T lymphocytes are MHC non-restricted NK cells, wherein tumor killing T cells (CD3+ CD8+ CTL cells) in the tumor-infiltrating lymphocytes are closely related to the clinical treatment effect of tumors.
At present, the culture of tumor infiltrating lymphocytes is mainly performed in a conventional two-dimensional culture environment, however, tumor killer T cells in the tumor infiltrating lymphocytes grow slowly and even die in the conventional two-dimensional culture environment, and the expansion efficiency of the tumor killer T cells in different culture batches is different. In addition, few specific antigens are currently identified for a particular tumor and cannot activate and maintain the lethality of tumor killer T cells expanded in vitro.
Disclosure of Invention
The purpose of the present disclosure is to provide a method for expanding tumor killer T cells in tumor infiltrating lymphocytes.
In order to achieve the above objects, the present disclosure provides a method for amplifying tumor killer T cells among tumor infiltrating lymphocytes, the method comprising the steps of:
a. mixing the monocyte suspension with the feed layer material suspension, and performing suspension culture for 96-120h to obtain a first culture; wherein the feeder layer material comprises 3D tumor organoids after irradiation inactivation;
b. mixing the first culture with a tumor infiltrating lymphocyte culture medium, and performing suspension culture for 65-72h to obtain a second culture;
c. mixing the second culture with the tumor infiltrating lymphocyte culture medium, and performing suspension culture for 72-96h to obtain a third culture;
d. removing the feeder layer material in the third culture, and separating to obtain the tumor killing T cells.
Optionally, in step a, the 3D tumor organoid is a 3D tumor organoid coated outside a microcarrier, wherein the microcarrier comprises microbeads with a diameter of 100-.
Optionally, the 3D tumor organoid is a particle with a particle size of greater than 100 μ ι η.
Optionally, in step a, the concentration of monocytes in said monocyte suspension is 1 × 106-5×106Per mL; the concentration of tumor cells in the feed suspension of the feeder layer is 0.5 × 106-5×106Per mL; the feeder material suspension is used in an amount of 1.2-2.0 parts by volume relative to 1 part by volume of the monocyte suspension.
Optionally, in steps b and c, the tumor infiltrating lymphocyte culture medium contains X-VIVOTM15 serum-free immune cell culture medium and 120-200IU/mL rhIL-2.
Optionally, in step b, the tumor infiltrating lymphocyte culture medium is used in an amount of 10-20 parts by volume relative to 1 part by volume of the first culture;
in step c, the tumor infiltrating lymphocyte culture medium is used in an amount of 40-50 parts by volume relative to 1 part by volume of the second culture.
Optionally, the suspension culture is performed at a stirring rate of 30-50 rpm/min.
Optionally, the preparation method of the feeder layer material comprises the following steps:
(1) culturing the 3D tumor organoid in a culture medium containing microcarriers to obtain a culture solution containing microcarriers coated with the 3D tumor organoid;
(2) and (3) performing irradiation inactivation on the culture solution containing the microcarrier coated with the 3D tumor organoid, then separating particles with the particle size larger than 100 mu m in the material subjected to irradiation inactivation, and taking the obtained particles as a feeding layer material.
Optionally, the microcarrier content in the microcarrier-containing culture medium is 1.0-2.2g/L, and the tumor cell content in the culture solution containing microcarriers coated with 3D tumor organoids is 1 × 109-5×109And (2) per liter.
Optionally, the irradiation dose for performing irradiation inactivation is 30-80Gy, and the irradiation inactivation time is 30-60 min.
By adopting the technical scheme, the in-vitro directional amplification of the tumor killing T cells can be realized, the amplification efficiency and the tumor killing activity of the tumor killing T cells are greatly improved, and the consistency of the amplification efficiency among different amplification batches can be improved.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Detailed Description
The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
The present disclosure provides a method for amplifying tumor killer T cells in tumor infiltrating lymphocytes, the method comprising the steps of: a. mixing the monocyte suspension with the feed layer material suspension, and performing suspension culture for 96-120h to obtain a first culture; wherein the feeder layer material comprises 3D tumor organoids after irradiation inactivation; b. mixing the first culture with a tumor infiltrating lymphocyte culture medium, and performing suspension culture for 65-72h to obtain a second culture; c. mixing the second culture with the tumor infiltrating lymphocyte culture medium, and performing suspension culture for 72-96h to obtain a third culture; d. removing the feeder layer material in the third culture, and separating to obtain the tumor killing T cells.
The inventor of the present disclosure finds that the tumor killing T cells in the tumor infiltrating lymphocytes are suspension cells with aggregation growth characteristics, and when performing the tumor killing T cell amplification culture, it is necessary to ensure that the tumor killing T cells are fully activated by the tumor specific antigen and fully contact with the growth factor, so as to ensure that the tumor killing activity and the amplification efficiency of the tumor killing T cells of different amplification batches are consistent. However, in the conventional two-dimensional culture environment, it is difficult for the tumor killing T cells growing in clusters to be in sufficient contact with the tumor specific antigen and growth factor present in the culture medium, and some of the tumor killing T cells grow slowly or even die because they are not sufficiently activated, so that there is a great difference in the tumor killing activity and the amplification efficiency of the tumor killing T cells of different amplification batches. The inventor of the present disclosure has tried to use a 3D tumor organoid cultured by a microcarrier after irradiation inactivation as a feeder layer material for tumor killer T cell expansion culture, and unexpectedly found that the feeder layer material can not only directionally expand tumor killer T cells, but also greatly improve the expansion efficiency and tumor killing activity of the tumor killer T cells, thereby obtaining the present disclosure.
By adopting the technical scheme, the in-vitro directional amplification of the tumor killing T cells can be realized, the amplification efficiency and the tumor killing activity of the tumor killing T cells are greatly improved, and the consistency of the amplification efficiency among different amplification batches can be improved.
The 3D tumor organoid disclosed by the disclosure is a 'micro tumor' obtained by three-dimensional culture of tumor cells, can simulate tumor tissues in structure and function, and can effectively maintain characteristics of tumor such as histology, immunology, gene mutation and the like. The tumor cells used for three-dimensional culture can be any tumor cells or tumor tissues known in the medical science at present, for example, tumor cell lines such as HepG2, H441 and HCT116, and tumor cells separated from tissues such as colorectal cancer, ovarian cancer, liver cancer operation or puncture tissues, lung cancer hydrothorax and ascites tissues and breast cancer bone metastasis.
In accordance with the present disclosure, 3D tumor organoids obtained using three-dimensional culture techniques can be used in the present disclosure, e.g., the 3D tumor organoids can be tumor organoids obtained using microcarrier culture; preferably, the 3D tumor organoid in step a is a 3D tumor organoid coated outside a microcarrier.
Wherein the type of the microcarrier can be selected in a wide range, for example, the microcarrier can be various microbeads with the diameter of 100-250 μm, which are suitable for anchorage-dependent cell growth; the microcarrier can be prepared from a natural scaffold material and/or an artificially synthesized scaffold material; the natural scaffold material may include, for example, collagen and/or dextran, and the synthetic scaffold material may include polylactic acid and/or chitosan; preferably, the vector is a HyQSpheres CGEN102-L type microcarrier. Wherein the HyQSpheres CGEN102-L type microcarrier is a product which is purchased from Thermo Fisher company and has the commercial number of SV 30044. The HyQSpheres CGEN102-L type microcarrier is a collagen-coated plastic microbead, has a large specific surface area, and due to the structure of the hard plastic microbead, the microcarrier in cells can be quickly separated and recovered through filtration.
Preferably, according to the present disclosure, the 3D tumor organoid may be a particle having a particle size of greater than 100 μm.
According to the present disclosure, preferably, in step a, the concentration of monocytes in the monocyte suspension may be 1 × 106-5×106Per mL; the concentration of tumor cells in the feed suspension of the feeder layer is 0.5 × 106-5×106Per mL; the feeder layer material suspension may be used in an amount of 1.2-2.0 parts by volume relative to 1 part by volume of the monocyte suspension.
Preferably, in step b and step c, the tumor-infiltrating lymphocyte culture medium contains X-VIVO according to the present disclosureTM15 serum-free immune cell culture medium and 120-200IU/mL rhIL-2.
According to the present disclosure, preferably, in step b, the tumor infiltrating lymphocyte culture medium is used in an amount of 10-20 parts by volume relative to 1 part by volume of the first culture; in step c, the tumor infiltrating lymphocyte culture medium is used in an amount of 40-50 parts by volume relative to 1 part by volume of the second culture.
According to the present disclosure, preferably, the suspension culture is performed at a stirring rate of 30-50 rpm/min.
According to the present disclosure, preferably, the preparation method of the feeder layer material comprises: (1) culturing the 3D tumor organoid in a culture medium containing microcarriers to obtain a culture solution containing microcarriers coated with the 3D tumor organoid; (2) and (3) performing irradiation inactivation on the culture solution containing the microcarrier coated with the 3D tumor organoid, then separating particles with the particle size larger than 100 mu m in the material subjected to irradiation inactivation, and taking the obtained particles as a feeding layer material.
Wherein the culture medium containing the microcarrier contains 5-20 mu M Rho kinase inhibitor Y27632, 100-250ng/mL basic fibroblast growth factor, 50-150ng/mL epidermal growth factor, 3-10 mu g/mL insulin, 30-60% Wnt-3a, 0.2-1.0g/mL bovine serum albumin and other growth factors and RPMI1640 culture medium.
According to the present disclosure, preferably, the microcarrier content in the microcarrier-containing culture medium is 1.0-2.2g/L, and the tumor cell content in the culture solution containing microcarrier coated with 3D tumor organoid is 1 × 109-5×109And (2) per liter.
According to the present disclosure, preferably, the irradiation dose for performing irradiation inactivation is 30-80Gy, and the irradiation inactivation time is 30-60 min.
The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby. The materials, reagents, instruments and equipment involved in the embodiments of the present disclosure may be obtained commercially, unless otherwise specified.
Examples
1. Preparation of 3D tumor organoids
1.1 microcarrier pretreatment
The pretreatment is carried out according to the specification of HyQSpherescCGEN 102-L type microcarrier (Thermo Fisher, SV30044), and the specific operation comprises the following steps: weigh 5g of dry microcarriers into siliconized glass bottles and use 200ml Mg free2+、Ca2+The PBS (200 mL per g of microcarrier) is soaked and expanded at room temperature for at least 20 minutes, and the supernatant is discarded; the microcarrier solution was then autoclaved (temperature 121 ℃, pressure 15psi, time 30 min). Discarding the liquid after high-temperature sterilization, and placing the liquid in a culture dish after siliconization for storage.
Before using the sterile microcarrier, 200ml of Mg-free microcarrier is used2+、Ca2+PBS (200 mL per g of microcarrier) was rinsed and the supernatant discarded, and the microcarriers were placed in 50mL of RPMI1640 medium containing growth factors such as 10. mu.M Rho kinase (ROCK) inhibitor Y27632(Sigma-Aldrich, MO), 200ng/mL basic fibroblast growth factor (bFGF), 100ng/mL Epidermal Growth Factor (EGF), 5. mu.g/mL insulin (insulin), 50% Wnt-3a, and 0.4g/100mL Bovine Serum Albumin (BSA), and equilibrated at 37 ℃ for 1 hour.
1.2 isolation of tumor cells from tissue samples
The postoperative tumor tissue of the solid tumor patient is soaked and preserved in a 20ml centrifuge tube of RPMI-1640 culture medium containing 0.5-1.5% vitamin C injection, and is transferred to a laboratory within 24 hours for aseptic experimental operation.
The tumor tissue was transferred into a 100mm petri dish using sterile blunt forceps and rinsed three times with PBS solution to remove blood clots. After rinsing was completed, 10ml of a 0.1% collagenase type II solution was added to the petri dish, the tumor tissue was minced with sterile scissors to form a minced meat, and the petri dish was placed in a 37 ℃ incubator for enzymatic hydrolysis for 1 hour. Sucking the cell enzyme solution, sieving with a 100-micron cell sieve, sucking into a centrifuge tube, placing in a 4-DEG C precooled centrifuge, centrifuging for 3 minutes at 300g, and discarding the supernatant. 15ml of feeder layer cell culture medium (RPMI-1640 medium containing 10% human AB serum) was added to the centrifuge tube, and the cell pellet was gently pipetted and resuspended for cell counting.
1.3 inoculation of tumor cells into microcarriers
At 1.0X 10 per ml5Concentration of tumor cells were inoculated in a medium containing 40 microcarriers/mL containing 10. mu.M Rho kinase (ROCK) inhibitor Y27632(Sigma-Aldrich, MO), 200ng/mL basic fibroblast growth factor (bFGF), 100ng/mL Epidermal Growth Factor (EGF), 5. mu.g/mL insulin (insulin), growth factors such as 50% Wnt-3a and 0.4g/100mL Bovine Serum Albumin (BSA), and RPMI1640 medium; the cells were stirred at 50rpm for 1 minute in an impeller-equipped cell culture flask and left to stand for 8 minutes in an intermittent stirring manner, and then cultured in a 5% carbon dioxide incubator at 37 ℃.
After 12-16 hours of culture, 200mL of RPMI1640 medium containing growth factors such as 200ng/mL basic fibroblast growth factor (bFGF), 100ng/mL Epidermal Growth Factor (EGF), 50% Wnt-3a and 0.4g/100mL Bovine Serum Albumin (BSA) was added to the medium and placed on a low speed mixer at 60rpm and placed in a 5% carbon dioxide incubator at 37 ℃. The culture was continued for 6 days with fluid replacement every 3 days.
1.43D tumor organoid Collection and processing
After the culture, the stirring of the culture flask was stopped, the microcarriers were allowed to settle naturally, 150ml of supernatant was discarded, 50ml of culture medium and 3D tumor organoids attached to the microcarriers were left, and irradiation inactivation was performed for 30 minutes using cesium 137 with a radiation dose of 50 Gray. Filtering and collecting the 3D tumor organoids after radiation inactivation by using a 100-micron filter membrane in a biological safety cabinet according to the content of 1 × 107The material of the 3D tumor organoids of individual tumor cells was dispensed as one portion and frozen stock solution containing 10% DMSO + 90% human AB serum was added as the feeder layer material.
2. Expanding tumor killer T cells
2.1 monocyte isolation
Collecting 50mL (heparin sodium anticoagulation) human peripheral blood, centrifuging in a centrifuge tube (800 Xg, 10min), taking supernatant for standby after inactivation and centrifugation of upper layer plasma, re-suspending lower layer precipitates of the blood with 20-30mL of physiological saline to obtain a physiological saline blood sample mixed solution, adding 15mL of lymphocyte separated fluid into a new centrifuge tube, wherein the density of the lymphocyte separated fluid is 1.077g/mL, slowly adding the physiological saline blood mixed solution to the upper layer (800g, 20min slow ascending and slow descending) of the lymphocyte separated fluid, centrifuging, taking a middle leucocyte layer, cleaning twice, obtaining mononuclear cells (PBMCs) and counting cells.
2.2 monocyte inoculation and culture
Recovering the above feeder layer material by conventional cell recovery method, centrifuging to remove frozen stock solution, transferring to WHEATON Magna Flex culture flask (IFNORS, 356834), adding 50ml X-VIVOTM 15 serum-free immunocyte culture medium (Lonza, 04-418Q), and making into culture medium containing 1 × 10 per ml6A mass of individual tumor cells was pre-warmed at 37 ℃ for 1 hour. Taking 10mL of X-VIVOTM 15 serum-free immune cell culture medium containing 200IU/mL rhIL-2 and pre-warming for 1 hour at 37 ℃, and re-suspending 1 part of mononuclear cells to ensure that the concentration of the mononuclear cells is 1 multiplied by 106Per mL; inoculating monocyte suspension into suspension containing 1 × 10 cells6WHEATON Magna Flex flasks (IFNORS, 356834) of 3D tumor organoid feeder layer material for individual tumor cells at 45rpm/mThe in-stirring rate maintains the suspension of the 3D tumor organoid feeder material to facilitate spatial contact of the monocytes with feeder cells. And on the third day of culture, 50mL of X-VIVOTM 15 serum-free immune cell culture medium containing 200IU/mL rhIL-2 is supplemented. The sixth day of culture, 100mL of X-VIVOTM 15 serum-free immune cell culture medium containing 200IU/mL rhIL-2 was supplemented. On day 9 of culture, cells were harvested and sampled for flow cytometry cell phenotype (CD3+ CD8+) and CCK8 assay to determine the killing rate of KCD3+ CD8+ cells on tumor organoid cells at a ratio of E: T of 1:1, and fold expansion was calculated.
Comparative example
The procedure was carried out as in example 1, except that the tumor cells isolated from the tumor tissue in example 1 were collected and irradiated as a feeder layer material containing 1.0X 10 cells/ml without 3D culture using microcarriers, using only two-dimensional culture tumor cells6The concentration of each tumor cell was 1.0X 106Mixing the mononuclear cells uniformly, directly adding 200mL of X-VIVOTM 15 serum-free immune cell culture medium containing 200IU/mL rhIL-2, placing the mixture on a WAVE Biorealtor system (GE, WAVE Biorealtor) for co-culture, shaking culture bottles at intervals of 45rpm/min once, after 9 days of culture, harvesting the cells, sampling flow-type detection cell phenotype (CD3+ CD8+) and CCK8 test for detecting the killing rate of KCD3+ CD8+ cells to tumor organ cells at the ratio of E: T being 1:1, and calculating the amplification multiple.
Test example
The procedures of examples and comparative examples were performed 20 times, respectively, and the amplification rate and purity of CD3+ CD8+ CTL cells activated by different tumor organoid amplifications and the average value of the killing activity of tumor organoid cells constructed from the surgical tissue of the corresponding patient were calculated, and the coefficient of variation (CV value, ratio of standard deviation to average value) of each index was calculated in 20 procedures, and the results are shown in Table 1.
TABLE 1
As can be seen from the data in table 1, the present disclosure can greatly improve the efficiency and activity of tumor killer T cells, while achieving high consistency among multiple amplification batches.
The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
Claims (10)
1. A method for expanding tumor killer T cells in tumor infiltrating lymphocytes, comprising the steps of:
a. mixing the monocyte suspension with the feed layer material suspension, and performing suspension culture for 96-120h to obtain a first culture; wherein the feeder layer material comprises 3D tumor organoids after irradiation inactivation;
b. mixing the first culture with a tumor infiltrating lymphocyte culture medium, and performing suspension culture for 65-72h to obtain a second culture;
c. mixing the second culture with the tumor infiltrating lymphocyte culture medium, and performing suspension culture for 72-96h to obtain a third culture;
d. removing the feeder layer material in the third culture, and separating to obtain the tumor killing T cells.
2. The method according to claim 1, wherein in step a, the 3D tumor organoid is a 3D tumor organoid coated outside a microcarrier, wherein the microcarrier comprises microbeads with a diameter of 100-250 μm, preferably the microcarrier is a HyQSpheres CGEN102-L type microcarrier.
3. The method of claim 2, wherein the 3D tumor organoids are particles having a size greater than 100 μ ι η.
4. The method of claim 1, wherein in step a, the concentration of monocytes in said monocyte suspension is 1 x 106-5×106Per mL; the concentration of tumor cells in the feed suspension of the feeder layer is 0.5 × 106-5×106Per mL; the feeder material suspension is used in an amount of 1.2-2.0 parts by volume relative to 1 part by volume of the monocyte suspension.
5. The method of claim 1, wherein in steps b and c, the tumor-infiltrating lymphocyte culture medium comprises X-VIVOTM15 serum-free immune cell culture medium and 120-200IU/mL rhIL-2.
6. The method of claim 5, wherein in step b, the tumor infiltrating lymphocyte culture medium is used in an amount of 10-20 parts by volume relative to 1 part by volume of the first culture;
in step c, the tumor infiltrating lymphocyte culture medium is used in an amount of 40-50 parts by volume relative to 1 part by volume of the second culture.
7. The method according to claim 1, wherein the suspension culture is performed at a stirring rate of 30-50 rpm/min.
8. The method according to any one of claims 1 to 3, wherein the feeder layer material is prepared by a method comprising:
(1) culturing the 3D tumor organoid in a culture medium containing microcarriers to obtain a culture solution containing microcarriers coated with the 3D tumor organoid;
(2) and (3) performing irradiation inactivation on the culture solution containing the microcarrier coated with the 3D tumor organoid, then separating particles with the particle size larger than 100 mu m in the material subjected to irradiation inactivation, and taking the obtained particles as a feeding layer material.
9. The method of claim 8, wherein the microcarrier is present in an amount of 1.0-2.2g/L in the microcarrier-containing culture medium, and the tumor cells are present in an amount of 1X 10 in the 3D tumor organoid-coated microcarrier-containing culture broth9-5×109And (2) per liter.
10. The method according to claim 8, wherein the irradiation dose for the inactivation by irradiation is 30-80Gy and the time for the inactivation by irradiation is 30-60 min.
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| CN113881630A (en) * | 2021-09-28 | 2022-01-04 | 创芯国际生物科技(广州)有限公司 | Method for culturing and separating tumor-specific TIL cells |
| WO2022143785A1 (en) * | 2020-12-31 | 2022-07-07 | Shanghai Cellular Biopharmaceutical Group Ltd. | Methods for preparing tumor-infiltrating lymphocytes |
| WO2022262805A1 (en) * | 2021-06-17 | 2022-12-22 | 上海赛比曼生物科技有限公司 | Method for preparing feeder cell bank |
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